dynamics and interaction of interleukin-4 receptor … dynamics 1 dynamics and interaction of...
TRANSCRIPT
IL-4R dynamics 1
Dynamics and interaction of Interleukin-4 receptor subunits in
living cells
Hetvi Gandhi, Remigiusz Worch, Martin Hintersteiner, Kristina Kurgonaite, Petra Schwille,
Christian Bökel & Thomas Weidemann
Supporting Material
Figures S1-S8
Tables S1-S3
IL-4R dynamics 2
FIGURE S1 HEK293T as a model for IL-4R mediated JAK/STAT signaling
FIGURE S1 Endogenous type 2 IL-4R signaling in HEK293T cells transiently transfected
with human STAT6 and assayed by phospho-STAT6 immunoblotting. At identical ligand
concentrations, IL-4 causes stronger activation than IL-13.
IL-4R dynamics 3
FIGURE S2 trisNTA coupled fluorescent dyes bind reversibly to hexahistidine tags
FIGURE S2 (A) FRET experiment: Using non-fluorescent trisNTA-QSY7 as an acceptor,
eGFP-H6 donor fluorescence was reversibly quenched. (B) Kinetics of the same experimental
procedure. Binding is completed in less than 5 minutes. All measurements were done at room
temperature in PBS using a Fluorolog τ-3 (Jobin-Yvon).
IL-4R dynamics 4
FIGURE S3 Co-expression of full length IL-4Rα chain with the type 2 receptor IL-
13Rα1
FIGURE S3 (A) Transiently expressed IL-13Rα1-eGFP shows efficient transport to the
plasma membrane with a mild accumulation in perinuclear membrane compartments at higher
expression levels. Cell morphology is indistinguishable from non-transfected cells. The
endogenous IL-4Rα chains are expressed at such a low density that cannot be visualized by
labeled IL-4 ligand (B). Ectopically expressed IL-4Rα chain can be detected by labeled IL-4-
A647 at the cell surface. However, a large fraction of cells transform into a rounded shape
with strong ER retention of the second receptor chain IL-13Rα1-eGFP. The morphology of
those cells is not suitable for single-cell FCCS analysis.
IL-4R dynamics 5
FIGURE S4 FCCS quantification of IL-4R subunit interactions in the plasma
membrane
FIGURE S4 (A) Histograms of the average cross-correlation ratio (CCav) from individual
cells were fitted by Gaussian distributions, and (B) their overlay for the indicated experiments
(color code). A shift towards higher CCav values indicates stronger binding. (C) Correlation
between the Kd values derived for single cells using either the eGFP (index G) or the A647
(index R) channel for normalization of the cross-correlation amplitude. The slope should be
close to one. (for details see the main article's appendix: "Kd determination from FCCS data").
IL-4R dynamics 6
FIGURE S5 FCCS analysis of type 2 IL-4R employing a His-tagged full length IL-4Rα
chain.
FIGURE S5 (A) Example for transformed, rounded HEK293T cells showing insufficient co-
expression of the two receptor subunits at the plasma membrane. (B) Exceptional case of
stretched cells showing good surface expression of both receptor subunits which were
amenable for FCCS measurements. Note their co-localization within cortical endosomes
(CEs, inset, arrow heads). (C) Cross-correlation amplitudes show a significant, ligand induced
increase indicating receptor subunit dimerization of the type 2 IL-4R complex under
overexpression conditions. Maximum cross-correlation (CCmax) reflects the number of dimers
with respect to the less abundant receptor subunit expressed in that particular cell (each data
points represents an average of 1-4 15 second FCCS runs). Cross-correlation levels were
normalized with a positive control (PC, H6-IL4Ram266-eGFP, dashed red line) which was
measured for each Labtek slide to combine independent experiments (n = 5). Box-and-
whisker plots indicate 2nd and 3rd quartile (box), mean (square), median (horizontal line),
and 1.5x interquartile range (whiskers). *** indicates p < 0.01 (ANOVA).
IL-4R dynamics 7
FIGURE S6 IL-4R subunits accumulate in cortical endosomes
FIGURE S6 HEK293T cells were transiently transfected with a non-tagged IL-2Rγ and
JAK3-eGFP to label CEs. Confocal images show the bottom membrane of a cell expressing
rather low amounts of JAK3-eGFP, therefore the background from unbound cytoplasmic
JAK3-eGFP is low. Dots of high intensity represent IL-2Rγ/JAK3-eGFP complexes enriched
in CEs. Comparing the fluorescence pattern at 0 min and 31.1 min reveals that their relative
positions are largely maintained. Smaller endosomes can show jump distances of a few µm
per minute (open arrow head), whereas larger endosomes move on a submicrometer length
scale (filled arrow head). Imaging was done at 22 °C in air buffer. The positions of the arrow
heads are fixed relative to the frames.
IL-4R dynamics 8
FIGURE S7 Cortical endosomes decorate with markers for early sorting and recycling
FIGURE S7 (A) CEs do not carry markers for late degradatory compartments. (B) IL-13Rα1
co-localizes with CEs marked by JAK3-tagRFP and H6-IL-2Rγ labeled with trisNTA-A647
(arrows) suggesting that type 1 and type 2 receptor subunits engage the same endosomal
trafficking route.
IL-4R dynamics 9
FIGURE S8 Constitutive internalization concentrates ligands and receptors within
cortical endosomes
FIGURE S8 (A) The truncated IL-4Ram266 chain promotes similar redistribution kinetics of
fluorescently labeled IL-4-A647 into CEs as the full length receptors. (B) Examples showing
cells before and after loading of trisNTA-A647. The amount of trisNTA-A647 (red) loaded
per CE (insets) was used for quantifying the kinetics. (C) Schematic workflow: Cells were
stained at 4°C, excess trisNTA-A647 was removed from the bulk, and endocytosis was
released by transfer to 37°C. The amount of receptor mediated trisNTA-A647 in CEs was
determined for different time points after the washing step by APD imaging.
IL-4R dynamics 10
TABLE S1: Receptor mobility determined by FCCS. Mean values for the indicated color
channels and constructs measured in HEK293T stained with trisNTA-A647: diffusion
coefficient of the fast component (fraction in %) in the A647 channel accounting for unbound
trisNTA-A647 in bulk solution (DR1), diffusion coefficient of the slow component in the
A647 channel reflecting lateral diffusion of trisNTA-A647 bound to H6-IL-4Ram266 in the
membrane (DR2), a short correlation time (fraction in %) measured in the eGFP channel (τ1),
diffusion coefficient of the slow component in the eGFP channel reflecting lateral receptor
diffusion of eGFP-tagged receptors in the membrane (DG2), diffusion coefficient of the cross-
correlation time (DX), and the size of sample (n). "n.d." indicates not determined. Mean values
in the grey shaded column correspond to Figure 3 D.
Experiment
DR1 ± SD
A647
[µm-2/s]
DR2 ± SD
A647
[µm-2/s]
τ 1 ± SD
eGFP
[ms]
DG2 ± SD
eGFP
[µm-2/s]
DX ± SD
[µm-2/s]
n
cells
H6- IL-4Rαm266-
eGFP
+trisNTA-A647
380 ± 220
(23 ± 7 %) 0.23 ± 0.07
0.70 ± 0.34
(24 ± 5 %) 0.24 ± 0.09 0.19 ± 0.08 55
Fas-eGFP 340 ± 210
(25 ± 6 %) 0.25 ± 0.06
0.76 ± 0.27
(29 ± 5 %) 0.42 ± 0.12 n.d. 8
Fas-eGFP + IL-4 590 ± 250
(31 ± 6 %) 0.31 ± 0.06
0.54 ± 0.16
(29 ± 5 %) 0.51 ± 0.13 n.d. 8
IL-2Rγ-eGFP / JAK3 450 ± 320
(25 ± 6 %) 0.25 ± 0.06
0.44 ± 0.15
(34 ± 10 %) 0.16 ± 0.05 n.d. 13
IL-2Rγ-eGFP / JAK3
+IL-4
410 ± 290
(23 ± 8 %) 0.23 ± 0.08
0.46 ± 0.24
(36 ± 6 %) 0.17 ± 0.08 0.08 ± 0.08 30
IL-13Rα1-eGFP 420 ± 270
(26 ± 10 %) 0.26 ± 0.10
0.62 ± 0.32
(32 ± 7 %) 0.38 ± 0.17 n.d. 25
IL-13Rα1-eGFP
+ IL-4
320 ± 170
(24 ± 6 %) 0.24 ± 0.06
0.73 ± 0.26
(32 ± 7 %) 0.27 ± 0.09 0.17 ± 0.11 30
IL-13Rα1-eGFP
+ IL-13
320 ± 150
(29 ± 7 %) 0.29 ± 0.07
0.74 ± 0.34
(31 ± 4 %) 0.29 ± 0.10 0.28 ± 0.23 39
IL-4R dynamics 11
TABLE S2: Receptor densities and binding data determined by FCCS. Numerical values
for the lateral affinities as mentioned in the text (Kd,2D), normalized average cross-correlation
ratio (CCav,n), surface density in the A647 channel specifying the amount of truncated, His-
tagged H6-IL-4Rαm266 chain per µm2 (Nr), surface density in the eGFP channel specifying
the second, eGFP-tagged receptor subunit (Ng), ratio of receptor densities averaged across the
measured cell population (Nr/Ng), and the sample size (n). Mean values in the grey shaded
columns correspond to Figure 3 E and 3 F. Note comparable expression levels of all
constructs.
Experiment Kd,2D ± SEM
[µm-2]
CCav,n ± SD
[%]
Nr ± SD
[µm-2]
Ng ± SD
[µm-2] Nr/ Ng
n
(cells)
H6- IL-4Rαm266-
eGFP
+trisNTA-A647
n.d. 100 ± 25 200 ± 100 220 ± 130 1.0 62
Fas-eGFP n.d. 4 ± 2 560 ± 500 390 ± 290 1.4 8
Fas-eGFP + IL-4 n.d. 6 ± 3 370 ± 180 210 ± 90 1.8 10
IL-2Rγ-eGFP / JAK3 n.d. 13 ± 10 270 ± 140 150 ± 100 2.1 16
IL-2Rγ-eGFP / JAK3
+IL-4 1000 ± 150 19 ± 11 260 ± 170 140 ± 70 1.8 32
IL-13Rα1-eGFP n.d. 11 ± 10 340 ± 180 170 ± 150 2.5 20
IL-13Rα1-eGFP
+ IL-4 180 ± 40 51 ± 11 420 ± 290 250 ± 180 1.9 25
IL-13Rα1-eGFP
+ IL-13 480 ± 90 29 ± 11 240 ± 180 220 ± 140 1.1 38
IL-4R dynamics 12
TABLE S3: Co-localization of JAK3/IL-2Rγ positive CEs with endocytosis markers.
Values of the grey shaded column were plotted in Figure 5 B.
Marker [%]
co-localization ± SD
n
(endosomes)
n
(cells)
mRFP-Rab5 96.1 1.8 955 13
EEA1 83.8 7.1 1640 18
mRCherry-Rab11 91.3 1.9 442 10
mRFP-Rab7 15.4 2.4 991 15
Lamp1 12.4 8.3 1209 14
Lysotracker 17.1 9.5 1078 14